Exogenous oxygen is required for prostanoid induction under brain ischemia as evidence for a novel regulatory mechanism

Previously, we and others reported a rapid and dramatic increase in brain prostanoids (PG), including prostaglandins, prostacyclins, and thromboxanes, under ischemia that is traditionally explained through the activation of esterified arachidonic acid (20:4n6) release by phospholipases as a substrate for cyclooxygenases (COX). However, the availability of another required COX substrate, oxygen, has not been considered in this mechanism. To address this mechanism for PG upregulation through oxygen availability, we analyzed mouse brain PG, free 20:4n6, and oxygen levels at different time points after ischemic onset using head-focused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy. The oxygen half-life in the ischemic brain was 5.32 ± 0.45 s and dropped to undetectable levels within 12 s of ischemia onset, while there were no significant free 20:4n6 or PG changes at 30 s of ischemia. Furthermore, there was no significant PG increase at 2 and 10 min after ischemia onset compared to basal levels, while free 20:4n6 was increased ∼50 and ∼100 fold, respectively. However, PG increased ∼30-fold when ischemia was followed by craniotomy of nonMW tissue that provided oxygen for active enzymes. Moreover, craniotomy performed under anoxic conditions without MW did not result in PG induction, while exposure of these brains to atmospheric oxygen significantly induced PG. Our results indicate, for the first time, that oxygen availability is another important regulatory factor for PG production under ischemia. Further studies are required to investigate the physiological role of COX/PG regulation through tissue oxygen concentration.

Previously, we (17)(18)(19)(20)(21) and others (22)(23)(24)(25) have demonstrated a rapid (within seconds) and dramatic (∼30 fold) increase in brain PG upon global ischemia modeled by decapitation.Acute PG increase under these conditions is traditionally explained through the activation of arachidonic acid (20:4n6) cascade (16,(26)(27)(28)(29).In this pathway, the ischemia-associated depletion of tissue O 2 and energy substrates leads to decrease in energy charge, which results in cytosolic calcium increase and activation of multiple calciumand kinase-dependent lipases and consequent 20:4n-6 release.The rate-limiting enzymes for PG synthesis, cyclooxygenase 1 and 2 (COX), and downstream synthases convert released 20:4n-6 to PG, causing a dramatic PG increase upon ischemia.However, this traditional mechanism for PG regulation under ischemia and other conditions does not account for another critical substrate for COX activity, O 2 .A limited number of studies indicate that the Michaelis constant (K m ) for COX by O 2 (the O 2 concentration at which COX activity is equal to half the maximal activity) is between 10 and 100 μM (30,31), close to the brain tissue free O 2 concentration of ∼50 μM as calculated from (32) after applying Henry's Law.These data indicate that small alterations in tissue O 2 concentrations might change PG production and may serve as another level of tissue COX activity regulation in addition to 20:4n6 release.Because O 2 depletion may precede lipase activation upon brain ischemia, we speculated that PG are not increased under brain ischemia.Further, we proposed that the previously observed PG increase is associated with craniotomy required for brain removal for analysis when O 2 becomes available for COX activity.
To test this mechanism, we analyzed mouse brain O 2 concentrations, free 20:4n6, and PG levels at baseline, 30 s, 2, and 10 min after ischemia onset using headfocused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy.We report a short (5.32 ± 0.45 s) O 2 half-life [T 1/2 (O 2 )] in the ischemic brain, with O 2 decreasing to undetectable levels (<10 nM) within 12 s of ischemia onset.During this time, we did not detect changes in free 20:4n6 or PG levels.At the increased duration of ischemia, free 20:4n6 levels were significantly (up to 100-fold) increased without significant PG increase compared to basal levels.However, PG increased ∼30-fold when ischemia was followed by craniotomy without MW, with a similar free 20:4n6 increase in both cases, indicating that 20:4n6 release is not the only factor required for 20:4n6 cascade activation.Moreover, craniotomy performed on nonMW mice under anoxic conditions did not result in PG induction in the ischemic brain, while exposure of these ischemic brains to atmospheric O 2 dramatically induced PG, excluding tissue damage contribution to the PG increase in nonMW tissue, or PG degradation under MW.
Together with our and others' previous reports on PG stability under MW conditions (18,23), our results indicate, for the first time, that the ischemia event does not increase brain PG concentration, and PG production might be regulated through O 2 availability.Further studies are required to validate the physiological and pathological role for COX activity regulation through tissue O 2 concentration.

Animals
This study was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and under an animal protocol approved by the University of North Dakota IACUC (protocols 2103-5).Thirty-three male C57BL/6 mice at 4-6 months of age were used for experiments.Mice were given a standard laboratory chow and water ad libitum.

Oxygen assay
To determine O 2 concentration, an O 2 microsensor probe (Unisense OX-10 microsensor, Aarhus Denmark) was inserted into the mouse cortex.Mice were anesthetized with ketamine (100 mg/kg) (Dechra Pharmaceuticals, Northwich, United Kingdom) and xylazine (10 mg/kg) (Covetrus, Portland, ME) and mounted in a temperature controlled (Kent Scientific, Torrington, CT) stereotaxic surgery device (Stoelting, Wood Dale, IL) to maintain mouse body temperature at 37 • C using a rectal probe.A 1 × 1 mm borehole in the cranium was used to insert a calibrated 10-μm tipped O 2 microsensor probe (Unisense OX-10 microsensor, Aarhus, Denmark) to record the cortex O 2 concentration, −1 mm DV, 2 mm ML, and −2 mm AP to bregma (33).Bregma and lambda were aligned as described in (33).Because the small (10 μm) probe diameter did not leave a visible trace in the brain sections, we were unable to directly confirm the probe position in the cortex.Thus, we inserted a pulled glass capillary filled with Evans blue dye at the same coordinates to visualize probe placement in a separate group of mice (supplemental Fig. S1).O 2 levels were allowed to stabilize for 10-20 min followed by cervical dislocation.Data were compiled and T 1/2 (O 2 ) was calculated using statistical software (GraphPad Prism 10, Boston, MA).Specifically, an asymmetric five-parameter regression analysis gave the best fit to the O 2 concentration dynamics and was used to calculate observed T 1/2 (O 2 ), while plateau followed by 1-phase decay regression analysis was used to calculate a splay from theoretical decay curve on Fig. 1A.A separate group of mice was used to collect brain tissue for PG analysis.

Mouse brain collection from ischemic and control groups under atmospheric O 2
For non-microwave irradiated (nonMW) ischemic animals (nonMW group), mice were anesthetized with isoflurane and euthanized by cervical dislocation.Brains were removed at 0.5, 2, and 10 min postmortem to model the corresponding durations of global brain ischemia, and then immediately frozen in liquid N 2 .Mice from the 2-and 10-min ischemia groups were placed on a 37 • C heating mat before brain collection to maintain post-mortem enzymatic activity.Brain removal from the cranial vault took 30 s.This brain collection method was commonly used in previous studies (17,22,34) and exposes metabolically active brain tissue to high atmospheric O 2 concentrations before tissue analysis.Another group of mice was exposed to microwave irradiation (MW) to denature enzymes in situ at 0 s (control group), 30 s, 2, and 10 min after cervical dislocation (ischemic MW group).Similar to the nonMW group, mice from the 2-and 10-min ischemia groups were placed on a 37 • C heating mat.The brains from the MW group were metabolically inactive when exposed to high O 2 levels before tissue analysis.Whole frozen brains were pulverized into a homogenous powder under liquid N 2 before analysis.

Mouse brain collection under anoxic conditions
Mice were anesthetized with isoflurane, euthanized by cervical dislocation, and immediately placed into a Bactrox hypoxic chamber (Sheldon Manufacturing, Cornelius, OR) flushed with N 2 gas to undetectable O 2 concentrations (<0.1% O 2 ) measured by a Cytocentric O 2 controller (BioSpherix, Parish, NY).The whole brain was collected at 30 s after cervical dislocation as described above, divided into two hemispheres.One brain hemisphere was pulverized into a homogenous powder under liquid N 2 and extracted inside the anoxic chamber, while another was processed under normal atmospheric O 2 .
The UPLC system consisted of a Waters ACQUITY UPLC pump with a well-plate autosampler (Waters, Milford, MA) equipped with an ACUITY UPLC HSS T3 column (1.8 μm, 100 Å pore diameter, 2.1 × 150 mm; Waters) and an ACUITY UPLC HSS T3 Vanguard precolumn (1.8 μm, 100 Å pore diameter, 2.1 × 5 mm; Waters).The column temperature was set at 55 • C and the autosampler temperature was set at 8 • C. LC solvent A consisted of acetonitrile:water (40:60) with 10 μM ammonium acetate and 0.025% acetic acid.Solvent B was acetonitrile:2-propanol (10:90) containing 10 μM ammonium acetate and 0.02% acetic acid.The flow rate was 0.3 ml/min for the duration of the run and the initial solvent B was 30%.At 0.1 min, %B was increased to 54% over 10 min, and then to 99% B over 10 min.99% B was held for 8 min and then returned to initial conditions over 0.5 min.The column was equilibrated for 2.5 min between injections.
Free 20:4n6 was quantified using a quadrupole time-offlight mass spectrometer (Q-TOF, Synapt XS, Waters) with electrospray ionization in negative mode.20:4n6 was monitored in the low energy channel at 303.2324 m/z and 20:4n6d 8 at 311.2826 m/z with a mass window of 0.02.Lock spray for mass correction (leucine enkephalin, 100 pg/μl) was infused at a rate of 10 μl/min.20:4n-6 was confirmed by coelution and quantified using a standard curve built against 20:4n6-d 8 internal standard (Cayman Chemical Co).MassLynx V4.1 software (Waters) was used for instrument control, acquisition, and sample analysis.Free 20:4n6 values are expressed as μmol per kg wet tissue weight (μmol/kgww).

PG analysis
Following pulverization, PG was extracted using a liquid/ liquid extraction method as previously described (18).Pulverized brain tissue (∼20 mg) was weighed into a Tenbroeck homogenizer containing 3 ml of 2:1 acetone:saline with PGE 2 d 9 (Cayman Chemical Co) as an internal standard.Samples were homogenized and transferred to silanized (Sigmacote, Sigma, St. Louis, MO) screw top tubes, and subjected to centrifugation to remove proteins.Following centrifugation, supernatants were transferred to a new silanized screw top tube.Extracts were washed three times with 2 ml of n-hexane to remove non-polar compounds, 20 μl of 2 M formic acid was added for acidification, and 2 ml of chloroform containing 0.005% BHT was added to extract PG.Chloroform extracts were concentrated under a stream of N 2 , transferred to silanized microinserts (MicroSolv, Leland, NC), dried under N 2 , and redissolved in 25 μl of 1:1 acetonitrile:water.Ten μl was injected onto UPLC-MS for analysis.UPLC-MS/MS analysis was performed on a Waters TQS MS in multiple reaction monitoring (MRM) mode with electrospray ionization operated in negative ion mode as previously described (19,37).
The UPLC system consisted of a Waters ACQUITY UPLC pump with a well-plate autosampler (Waters) equipped with an ACUITY UPLC HSS T3 column (1.8 μm, 100 Å pore diameter, 2.1 × 150 mm; Waters) and an ACUITY UPLC HSS T3 Vanguard precolumn (1.8 μm, 100 Å pore diameter, 2.1 × 5 mm; Waters).The column temperature was set at 55 • C and the autosampler temperature was set at 8 • C. LC solvent A consisted of water with 0.1% formic acid and solvent B was acetonitrile with 0.1% formic acid with a flow rate of 0.45 ml/ min.Initially, solvent B was held at 39% for 0.5 min, then increased to 40.5% over the next 6.88 min, and finally increased to 98% over 0.2 min 98% B was held for 7 min and returned to initial over 0.2 min.The column was equilibrated for 2 min between injections.
PG were quantified using PGE 2 d 9 as an internal standard that has been previously validated as an internal standard for all prostanoids analyzed (18).PG were monitored in MRM mode with the following mass transitions as previously described ( 37

Statistical analysis
Statistical analysis was performed using GraphPad Prism 10 (GraphPad, Boston, MA).Statistical significance was determined by one-way ANOVA with the Tukey post hoc test.Values were considered significant with P value <0.05 and Brain PG regulation through oxygen availability are expressed as mean ± SD.T 1/2 (O 2 ) was calculated using nonlinear regression in GraphPad Prism 10 as described above.

Cortical oxygen levels during global ischemia
To quantify brain O 2 during ischemia, we used a microsensor O 2 probe (Unisense OX-10) inserted into the cortex.The small, 10 μm diameter tip of this probe allows for a real-time, tissue-specific free O 2 level measurement in the living animal with minimal mechanical damage and hypoperfusion due to reduced vascular compensation, and minimal O 2 consumption by the probe.Free O 2 levels were rapidly (within 12 s) depleted after the ischemia onset with T 1/2 (O 2 ) of 5.32 ± 0.45 s (Fig. 1A).There was a splay from the theoretical exponential decay curve (4 s, Fig. 1A) that might be explained by the diffusion latency of O 2 between the extracellular fluid where O 2 was measured, and the cellular fluid where O 2 was fast consumed upon ischemia.Thus, it is possible that the cellular T 1/2 (O 2 ) is shorter than the reported values.Consistent with previous reports (38,39), the basal cortical O 2 levels were 54.6 ± 3.2 μM (Fig. 1B).These data indicate that O 2 levels fall below Km(O 2 ) values of COX1/2 (in the 10 μM range (30,31,40,41)) within seconds of global ischemia onset.

PG production during ischemia
Next, we determined if previously reported rapid PG production following global brain ischemia (17)(18)(19)(20)(21)(22)(23)(24)(25) is associated with metabolically active tissue exposure to atmospheric O 2 during tissue removal from the cranium, rather than production in situ in the ischemic brain.In these experiments, we measured brain PG production at increasing ischemia durations followed by MW fixation to heat denature enzymes in situ (true PG levels without exogenous atmospheric O 2 contribution to ischemic brain metabolome) and without MW (exogenous atmospheric O 2 contribution to PG levels).Following global ischemia, we did not detect alterations in PG levels after in situ enzyme inactivation by MW (MW group) when compared to basal brain PG levels (MW, 0 min ischemia) (Fig. 2).However, at all time points after global ischemia, PG were significantly increased when metabolically active brains were exposed to atmospheric O 2 by removal from the cranium without MW (nonMW group, Fig. 2).Compared to the MW group, PGE 2 levels in nonMW brain tissue were increased 23-, 53-, and 109-fold at 0.5, 2, and 10 min of global ischemia, respectively.Similarly, we did not detect alterations in PGD 2 , 6-ketoPGF 1α , PGF 2α , and TXB 2 levels after MW when compared to basal brain PG levels (Fig. 2).Likewise, PGD 2 (63-, 226-, 544-fold), 6-ketoPGF 1α (20-, 55-, 143-fold), PGF 2α (46-, 104-, 198-fold), and TXB 2 (36-, 69-, 182-fold) levels at 0.5, 2, and 10 min, respectively, are all significantly increased following exposure to atmospheric O 2 without MW (Fig. 2).These data indicate that PG are not produced upon global brain ischemia as previously reported, but are instead produced when enzymatically active brain tissue is exposed to exogenous O 2 .

Non-esterified 20:4n6 levels during global ischemia
Because 20:4n6 is an essential substrate for COX, and 20:4n6 availability is considered to be the major regulatory mechanism for PG production under different conditions, including brain ischemia (16,(26)(27)(28)(29), we determined free 20:4n6 dynamics during brain ischemia, as well as the effect of MW on 20:4n6 levels at basal and ischemic conditions.MW did not affect free 20:4n-6 levels at 2 and 10 min when compared to the nonMW time counterpart (Fig. 3), which did not correlate with PG dynamics (Fig. 2).However, at 0.5 min, 20:4n6 level in nonMW brain was significantly increased compared to basal and MW 0.5 min 20:4n6 levels (13-and 11-fold, respectively).We speculate that this increase in free 20:4n6 is a result of mechanical damage during craniotomy and not due to phospholipase activity during extraction, or 20:4n6 degradation under MW conditions, as 20:4n6 levels in nonMW and MW at 2 and 10 min of ischemia were not significantly different from their time point counterpart.Consistent with this speculation, previous research shows that traumatic injury increases PLA 2 -mediated 20:4n6 release in the brain (42,43).Importantly, while MW and nonMW 20:4n6 levels are unchanged at 2 and 10 min of ischemia compared to their respective time point, PG production is significantly increased in nonMW brains (Fig. 2).This suggests that while free 20:4n6 levels are increased during global ischemia, availability of O 2 is crucial for PG production.Thus, the rapid depletion of free O 2 in the ischemic brain precedes 20:4n6 release and therefore PG production does not proceed in the intact cranial vault.

PG are not increased in the ischemic nonMW brains handled under anoxic conditions
To further confirm that exogenous atmospheric O 2 rather than ischemia or brain injury during craniotomy contributes to PG induction, and to bolster findings that MW does not destroy endogenous PG that are potentially produced during ischemia, we collected ischemic (0.5 min) nonMW brains under an anoxic environment.There was no difference in PG levels between MW tissue and nonMW tissue removed and extracted under anoxia (Fig. 4), suggesting that prostanoids are not induced in these injured ischemic samples, and MW does not destroy endogenous PG. Interestingly, when nonMW brains are collected under anoxic conditions but pulverized and extracted under atmospheric O 2 , there is a robust and significant increase in PG (Fig. 4).These data suggest that increased PG in nonMW ischemic brain samples (Fig. 2) is likely a result of PG production during sample preparation due to exposure to atmospheric O 2 , but not the result of brain injury per se or an artifact of MW.Thus, O 2 availability at any step during nonMW sample handling post-craniotomy increases PG levels.

DISCUSSION
In the present study, we addressed, for the first time, a novel mechanism for PG upregulation through O 2 availability.We report that none of the measured PG are increased in the ischemic brain, and the previously reported rapid and dramatic PG upregulation upon brain ischemia (17)(18)(19)(20)(21)(22)(23)(24)(25) is the result of brain tissue exposure to atmospheric O 2 during tissue removal from the skull.Considering the relatively low affinity of COX to O 2 , the described mechanism for oxygen-dependent PG production might have a significant role in PG regulation under other pathological or physiological conditions.
Previously, one study (23) demonstrated a significant 8-fold increase in TXB 2 concentration in the MW ischemic brain with little endogenous PGE 2 /D 2 induction, while other PG were not assayed in this study.The difference between TXB 2 and PGD 2 formation was explained through a different coupling between COX and downstream PG synthases.In the present study, we demonstrated, for the first time, no increase in any of 5 major COX-dependent products of 20:4n6 metabolism under brain ischemia including TXB 2 , and an O 2dependent mechanism for PG formation in the brain.To the best of our knowledge, no previous studies have addressed the relation between O 2 concentration and Brain PG regulation through oxygen availability PG production in the ischemic brain.It is difficult to speculate regarding previously reported TXB 2 increase in the ischemic intact brain (23), a finding which was not reproduced in the present study.It is possible that differences in sample handling or processing might contribute to the discrepancy in the results.For example, the decapitation used by (23) allows the injured tissue to be exposed to O 2 , thus maintaining some COX activity, while cervical dislocation used in the present study prevents this artifact.
Traditionally, a rapid PG increase upon ischemic onset is explained through an activation of 20:4n6 cascade (16,(26)(27)(28)(29).In this pathway, an ischemia event leads to tissue energy charge decrease, thus leading to the suppression of ATP-dependent ion pumps and consequent cytosolic Ca 2+ increase with activation of Ca-and kinase-dependent phospholipases.Activated phospholipases release 20:4n6 as a substrate for COX.
The product of COX activity, PGG 2 is then converted to PGH 2 and later, to distinct end-products of PG by downstream enzymes.
However, this traditional view on PG upregulation does not consider the second essential COX substrate, O 2 , as two molecules of O 2 are required for the cyclooxygenase reaction (44,45).It is well established that in the ischemic brain, O 2 decrease leads to energy deprivation and suppression of ATP-dependent ion pumps.Thus, O 2 decrease precedes ion disbalance, including cytosolic Ca 2+ increase (46), and might precede Ca-and kinase-dependent PLA 2 activation required for 20:4n6 release for PG synthesis.To this end, it is important to establish the exact dynamics of brain O 2 concentration during ischemia and estimate if COX is still active under O 2 concentrations in the ischemic brain when 20:4n6 is released.
In order to accurately measure mouse cortex O 2 concentration alteration under global ischemia in the present study, we used a small, 10 μm diameter O 2 electrode to prevent tissue damage and possible hypoperfusion from surrounding tissue compression and to minimize O 2 consumption by the electrode.Our data indicate a rapid decrease in O 2 from 54 ± 3 μM to undetectable levels (<10 nM) within 12 s of ischemia onset with the calculated T 1/2 (O 2 ) 5.32 ± 0.45 s (Fig. 1).However, it is possible that the cellular T 1/2 (O 2 ) is even shorter than the reported values because we observed a 4 s splay from theoretical exponential decay curve (Fig. 1A).The splay might be the result of diffusion latency of O 2 from the extracellular fluid where O 2 was measured, to the cellular fluid where O 2 was fast consumed upon ischemia.Consistent with the expected delay in PLA 2 activation discussed above, we did not detect free 20:4n6 changes after the first 30 s of ischemia (Fig. 3).At 2 and 10 min of ischemia, we detected a significant (over 100-fold) increase in free 20:4n6 (Fig. 3) without any significant increase in PG (Fig. 2), which is consistent with the absence of PG synthesis in the ischemic brain when O 2 concentration falls significantly below Km(O 2 ) values in the 5-10 μM range.In addition, metabolically active tissue harvested and processed under anoxic conditions did not increase PG levels, while the same tissue exposed to atmospheric O 2 dramatically increased all measured PG (Fig. 4).These data indicate, for the first time, that O 2 depletion precedes 20:4n6 release upon brain ischemia, and PG are not produced in the ischemic brain.This mechanism does not exclude the role for 20:4n6 release in PG regulation in the presence of sufficient O 2 concentrations.In this regard, liberated free 20:4n6 is readily available for PG formation in the ischemic tissue when O 2 concentration raises above Km(O 2 ), and explains a burst PG induction in the ischemic brain upon exposure to atmospheric O 2 (Figs. 2, 4).Our data indicate that O2 availability is an additional mechanism to regulate PG production that still requires 20:6n6 liberation.However, a number of artifacts and alternative mechanisms might contribute to the absence of PG upregulation in the ischemic tissue reported in the present study.First, for the samples where no PG upregulation was detected, we used MW to heat denature enzymes for PG synthesis in situ before tissue exposure to atmospheric O 2 (Fig. 2).Therefore, it is reasonable to consider that PG, that are known for their instability, were degraded or trapped in denatured protein upon MW, thus contributing to the lower levels observed in MW tissue.We and others have previously addressed this concern by measuring recovery of the endogenously induced PG after MW (18), or the recovery of exogenous labeled PG injected into tissue before and after exposure to MW (23,24).These studies clearly demonstrated that MW does not alter tissue PG levels and this is a safe "gold standard" method to prevent PG alterations during analysis.
Another consideration is the contribution of tissue injury to the observed difference in PG levels between ischemic MW and ischemic nonMW brains.MW samples were fixed before brain removal from the cranium, thus no injury was induced in these brains before the deactivation of enzymes.However, in nonMW ischemic samples, enzymes were active during brain removal from the cranium, causing injury-like damage which might account for additional PG upregulation in the ischemic samples.To test this mechanism, we analyzed nonMW brains removed from the cranium and extracted under anoxic conditions in a glove box.We did not detect PG upregulation in these "injured" samples (Fig. 4).However, when the same samples were exposed to atmospheric O 2 during sample pulverization and extraction, PG were significantly upregulated (Fig. 4).Importantly, PG are actively synthesized during sample preparation and extraction procedures when the tissue powder is exposed to atmospheric O 2 (18), thus PG are further upregulated during sample preparation and PG upregulation in nonMW tissue does not require instantaneous O 2 diffusion into the brain tissue during removal from the cranium.Together, these data confirm that O 2 availability but not tissue injury on its own is responsible for PG upregulation in nonMW brains and that PG are not upregulated in the ischemic brain.
In addition, we cannot exclude the effect of anesthesia on PG metabolism.In the present study, we used Brain PG regulation through oxygen availability ketamine/xylazine for O 2 sensor insertion since, in contrast to isoflurane, it does not require inhalation of O 2 to keep animals under anesthesia during the surgery, eliminating a possible artifact from brain hyperoxia when isoflurane is used to measure brain O 2 dynamics.However, we used isoflurane to briefly anesthetize mice before cervical dislocation and subjected them to MW to be consistent with our previous reports on brain PG metabolism (15,19,21).To address a possible effect of anesthesia on brain T 1/2 (O 2 ), O 2 dynamics were measured under both anesthetics upon ischemia.Under isoflurane, brain T 1/2 (O 2 ) was 3.58 ± 1.47, not significantly different from ketamine/xylazine (5.32 ± 0.45 s, Fig. 1), indicating that the form of anesthesia does not significantly change brain O 2 dynamics under ischemia.Importantly, anesthesia also affects brain fatty acid metabolism under basal and ischemia conditions.Deep pentobarbital anesthesia reduces palmitate turnover in brain phospholipids (50), and attenuates 20:4n6 liberation during global ischemia (51), though the effects of isoflurane or ketamine anesthesia have not been reported.It is reasonable to expect that anesthesia also affects PG production which depends upon 20:4n6 liberation.However, because all control and experimental animal groups used for PG analysis were subjected to the same anesthesia conditions, it is unlikely that anesthesia contributed to the observed differences in PG alterations between MW, nonMW, and N 2 -exposed brains.
Our reported findings might have an implication for other models and conditions such as injury and neuroinflammation.There is increasing evidence that PGinduced neuroinflammation contributes to clinical outcomes in traumatic brain injury (TBI), as reviewed in (52).Our data suggest that confounding differences in TBI models (closed vs. open head injury) could be exacerbated by varying levels of O 2 availability during trauma, contributing an additional level of PG regulation that might also be applied to clinical outcomes of human TBI or brain surgery.Additionally, our results indicate that the depth of O 2 penetration into the injured tissue, as well as the degree of tissue oxygenation before the injury, might be related to the difference in oxylipin production in different brain regions, with the highest level in the cortex and lowest in the brainstem (53).
Importantly, brain O 2 concentrations in some brain regions are within the COX Km(O 2 ) values and range from 1 μM O 2 in pons to 55 μM in cortex to 88 μM in pia in rats (38), with similar values in cats and rabbits (54), and 60 μM in the mouse somatosensory cortex (39).Therefore, it is reasonable to speculate that changes in the brain O 2 concentrations associated with changes in brain activity or pathological conditions might significantly alter PG production, especially in regions with low O 2 levels, thus positioning COX-PG as an O 2 sensor.Together with a well-established vasoactivity of PG (55,56) and their modulatory effect on neuron activity (57,58), O 2 -dependent PG regulation might have an important role in rapid brain adaptation to changes in energy and O 2 demands.
Though not challenged in the present study, other bioactive products of 20:4n6 and other polyunsaturated fatty acids oxidation, including LOX-dependent metabolites and non-enzymatic products of oxidation which have an important role under brain ischemia (53), might be regulated by the same mechanism.This speculation is supported by the similar COX Km(O 2 ) values reported for various lipoxygenases that are in the 8-26 μM range (40).Further studies are needed to address this possibility.
In summary, we demonstrated, for the first time, that PG upregulation is highly dependent upon O 2 availability.The described mechanism for O 2 -dependent PG production which might have a significant role in PG regulation under other pathological or physiological conditions, requires further investigation.cyclooxygenase 1 and 2; K m , Michaelis constant; MW, microwave irradiation; nonMW, non-microwave irradiated; PG, prostanoids; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2α, prostaglandin F2α; TXB2, thromboxane B2; UPLC-MS, ultra-high-pressure liquid chromatographymass spectrometry.

Fig. 1 .
Fig. 1.Oxygen level is rapidly depleted following global ischemia.Mice were fixed in a stereotaxic frame followed by oxygen microsensor insertion into cortex.Global ischemia was induced by cervical dislocation.A: Normalized cortex oxygen levels used to calculate O 2 half-life (T 1/2 ).Values are mean ± standard deviation (n, number of animals, =3) with the individual absolute value presented in panel B. T 1/2 was calculated using nonlinear regression in GraphPad Prism 10 as described in the Materials and Methods.B: Absolute levels of cortex O 2 before (54.6 ± 3.2 μM) and during global ischemia.

Fig. 2 .
Fig. 2. Prostanoids are not produced in the ischemic brain.Global brain ischemia was modeled by cervical dislocation.After cervical dislocation, brains were removed from the cranial vault either after microwave irradiation to heat denature enzymes in situ (MW) or collected without microwave irradiation (nonMW) at 0.5, 2, and 10 min postmortem.The MW group represents true PG levels in the ischemic and control brains without the artificial contribution of exogenous atmospheric O 2 during tissue handling, while the nonMW group represents PG levels affected by atmospheric O 2 during tissue handling.Postmortem mouse body temperature was maintained at 37 • C by wrapping mice with a temperature-controlled heating pad to maintain enzymatic activity following cervical dislocation.Basal prostanoids (PG) levels were determined in MW brain tissue without cervical dislocation (0 min of global ischemia).PG were quantified by UPLC-MS against stable isotope labeled internal standard.Values are mean ± standard deviation (n = 3-4) with individual values.Values that do not share the same letter are statistically different (P ˂ 0.05, one-way ANOVA with Tukey's post hoc test).

Fig. 3 .
Fig. 3. Brain free arachidonic acid (20:4n6) levels are unaffected by microwave irradiation during global ischemia.Mice were either microwave irradiated to inactivate enzymes (MW), or collected without microwave irradiation (nonMW) at 0.5, 2, and 10 min postmortem.Basal free 20:4n6 levels were determined in MW brain tissue at 0 min of global ischemia.Free 20:4n6 levels were determined by UPLC-MS.Values are mean ± standard deviation (n = 3-4) with individual values.Values that do not share the same letter are statistically different (P ˂ 0.05, one-way ANOVA with Tukey's post hoc test).

Fig. 4 .
Fig. 4. Prostanoids are not increased in the ischemic nonMW brains handled under anoxic conditions.Mice were subjected to 0.5 min global ischemia by cervical dislocation and brains were collected either after microwave irradiation to quench postmortem metabolism (MW, black bars) or collected without MW (nonMW) under anoxia in the anoxic chamber.After freezing in liquid nitrogen, one hemisphere was pulverized to a homogenous powder, and extracted in the anoxic chamber (white bars), while another hemisphere from each mouse was removed from the anoxic chamber and processed under atmospheric O 2 (gray bars).Prostanoid levels were determined by UPLC-MS.Values mean ± standard deviation (n = 3-4) with individual values.Values that do not share the same letter are statistically different (P ˂ 0.05, one-way ANOVA with Tukey's post hoc test).